Neural crest stem cells are a multipotent cell population arising at the neural plate border of neural ectoderm between the neural plate and the non-neural ectoderm during vertebrate embryogenesis (1, 2). As neural crest cells delaminate from the roof plate upon closing of the neural tube, they migrate throughout the body where they contribute to the peripheral nervous system, connective and skeletal cranial tissues, melanocytes and valves of the heart. Specification of ectoderm into neural, neural plate border and epidermal cells is directed by overlapping but distinct combinations of signaling molecules centering around Wnt, BMP and Fgf pathways (3-10). Knowledge of these pathways has been instrumental in establishing conditions for differentiation of human pluripotent cells in culture along a neuroectoderm pathway for the generation of neural progenitor cells (NPCs) and a wide-range of neuronal sub-types (11-13)
Efficient methods for generation of NPCs from human pluripotent cells have recently been made possible by use of specific inhibitors, such as Noggin and SB 431542, that function by blocking BMP and Activin A/Nodal signaling, respectively (12). Simultaneous inhibition of these pathways is sufficient to drive pluripotent cells in culture down the neuroectoderm pathway, generating a population of Pax6+ Sox1+ Sox2+ NPCs that can assemble into neural rosettes as columnar epithelia. When isolated from neural rosettes in culture, NPCs can be amplified due to their self-renewing capacity and further differentiated into a wide range of neural cell types (13, 14). Neural crest cells are typically found interspersed with neural rosettes in such cultures and can only be obtained as a highly enriched cell population by cell sorting techniques (15, 16). Alternative methods for generating neural crest cells from pluripotent cells have been described, but these utilize co-culture on feeder layers (16, 17), are relatively inefficient and also require cell sorting to generate highly enriched populations. These methods all involve complex, multistep procedures that yield relatively low yields of p75+ Hnk1+ neural crest. These issues highlight the limitations of current approaches and consequently, severely limit their utility in scale-up applications for tissue engineering, regenerative medicine and drug screening applications. An efficient, single-step method for generation of neural crest cells from pluripotent cells would therefore represent a significant advancement in understanding their biology and towards their biomedical application.
The heterogeneity of neuroectoderm cultures from which neural crest cells are currently isolated led us to reevaluate the general approach from a cell signaling perspective. While low levels of BMP and Activin A signaling are considered pre-requisites for the generation of NPCs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the potential role of Wnt in specifying neural crest has not been evaluated. This is somewhat surprising considering the well-established role for canonical Wnt signaling in neural crest development in vivo (3-5).